Sexual Reproduction and Seasonality of the Alaskan Red Tree Coral, pacifica

Rhian G. Waller1*, Robert P. Stone2, Julia Johnstone3, Jennifer Mondragon4 1 University of Maine, School of Marine Sciences, Darling Marine Center, Walpole, Maine, United States of America, 2 Alaska Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Juneau, Alaska, United States of America, 3 University of Maine, Darling Marine Center, Walpole, Maine, United States of America, 4 Alaska Regional Office, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Juneau, Alaska, United States of America

Abstract The red tree coral is an important habitat forming octocoral in North Pacific waters. Given the prominence of this species in shelf and upper slope areas of the Gulf of Alaska where fishing disturbance can be high, it may be able to sustain healthy populations through adaptive reproductive processes. This study was designed to test this hypothesis, examining reproductive mode, seasonality and fecundity in both undamaged and simulated damaged colonies over the course of 16 months using a deepwater-emerged population in Tracy Arm Fjord. Females within the population developed asynchronously, though males showed trends of synchronicity, with production of immature spermatocysts heightened in December/January and maturation of gametes in the fall months. Periodicity of individuals varied from a single year reproductive event to some individuals taking more than the 16 months sampled to produce viable gametes. Multiple stages of gametes occurred in polyps of the same colony during most sampling periods. Mean oocyte size ranged from 50 to 200 mm in any season, and maximum oocyte size (802 mm) suggests a lecithotrophic larva. No brooding larvae were found during this study, though unfertilized oocytes were found adhered to the outside of polyps, where they are presumably fertilized. This species demonstrated size-dependent reproduction, with gametes first forming in colonies over 42-cm length, and steady oocyte sizes being achieved after reaching 80-cm in length. The average fecundity was 86 (612) total oocytes per , and 17 (612) potential per polyp fecundity. Sub-lethal injury by removing 21–40% of colony tissue had no significant reproductive response in males or females over the course of this study, except for a corresponding loss in overall colony fecundity. The reproductive patterns and long gamete generation times observed in this study indicate that recruitment events are likely to be highly sporadic in this species increasing its vulnerability to anthropogenic disturbances.

Citation: Waller RG, Stone RP, Johnstone J, Mondragon J (2014) Sexual Reproduction and Seasonality of the Alaskan Red Tree Coral, Primnoa pacifica. PLoS ONE 9(4): e90893. doi:10.1371/journal.pone.0090893 Editor: John Murray Roberts, Heriot-Watt University, United Kingdom Received October 11, 2013; Accepted February 6, 2014; Published April 25, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was funded by the National Oceanic and Atmospheric Administration Alaska Fisheries Science Center, and through the National Geographic Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction otherwise we know very little that will assist us in gauging their capability to recover from disturbance. Data on the reproductive is one of the most dominant gorgonian families in processes of cold-water corals is still in its infancy compared to the deep-sea and polar regions, with 233 valid species known to date wealth of data available for their shallow water zooxanthellate [1]. Primnoids are found in all ocean basins and are most counterparts [10,9,11]. Advances in this discipline are largely numerous in Antarctic waters where the family is thought to have hampered by inability to sample specimens at regular intervals in originated [1]. This family is primarily deep-sea, with most current most deep-sea environments. records coming from slope and abyssal depths whereas shallow Recent submersible observations in the eastern Gulf of Alaska populations are rare. The Primnoa is only found in the North shelf habitats clearly indicate that red tree corals are highly Pacific, North Atlantic and South Pacific regions, vulnerable to disturbance from fishing gear and that a much with the red tree coral, Primnoa pacifica Kinoshita, 1907, known higher proportion of large colonies are damaged compared to only from North Pacific waters [2]. This species can attain massive smaller colonies. Some cold-water corals subjected to mechanical size (up to 5 m in height and 7 m in width; 3, R. Stone, personal disturbance exhibit reduced reproductive fitness (i.e. fecundity) via observations) and form vast monospecific thickets that provide resource re-allocation and planulae expulsion [12,13]. Further- habitat for fish [3,4] and invertebrates [5]. more, larger, more susceptible, colonies are thought to be Growth rates show that P. pacifica is a slow growing coral [6,7] considerably more fecund than smaller colonies [14]. Both of and there is an indication that successful recruitment events occur these factors may influence the ability of habitats dominated by on a decadal scale (Stone pers obs). Many cold-water gorgonians red tree corals to recover from fishing gear disturbance. appear to be gonochoristic (i.e. separate sexes at the colony level) Shallow-water habitats in several high-latitude fjords sustain brooders that likely have seasonal reproductive cycles [8,9] but benthic communities that are similar to those usually found in

PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e90893 Red Tree Coral Reproduction much deeper water. The term ‘‘deepwater emergence’’ has been Sample Collection used to describe this phenomenon, though the environmental Thirty-eight healthy (i.e. undamaged) colonies (42 to 162 cm factors that allow these species to survive much shallower than total length along the longest axis) were haphazardly selected for their usual distribution (often by thousands of meters) are not yet tagging between 8th and 10th September 2010. Colonies were fully understood. Shallow-water temperatures in these fjords tend tagged above the holdfast with numbered, plastic cinch-type tags to be much cooler mimicking temperatures usually found at by scuba divers. The depth range for all tagged colonies was 9.8 to bathyal depths [15,16]. Nutrient-rich upwelling, strong current 17.1 m mean lower low water (all depths reported hereafter are regimes, temperatures at the surface layers being equal to the deep referenced to mean lower low water). Reference images were ocean and reduced light levels are other factors that could collected of each colony with a meter stick scale, and Image J influence how these deep-sea species survive shallower than their (National Institutes of Health) was used to calculate flat-plane area normal distributions [7,16]. Several shallow systems worldwide of colonies. that support deepwater emerged coral species have been Samples were collected from all 38 tagged-colonies during six documented – Chile [17], Norway [18], New Zealand time periods over an 18-month span: September 2010, December [19,20,21], Alaska [7] and British Columbia [22]. The coral 2010, March 2011, June 2011, and January 2012. In September species that appear in these fjord systems are often ecosystem 2011, extremely poor visibility limited us to collected samples from engineers (in sensu [23]), altering both the local benthic and only 10 colonies (9 females and 1 male). The poor water visibility physical environment and providing suitable microhabitat for was due to heavy sediment loading and corresponded with warm colonization of many associated species [24]. These shallow-water air temperatures and the onset of the rainy season in the region— communities appear to mimic those of the deep sea and thus both factors increasing stream run-off and glacial melt. provide a unique opportunity to use the easily accessible habitats Pieces approximately 4–7 cm in length (containing ,30 polyps) as ‘‘living laboratories’’ to study the physiological processes of were carefully sampled from a distal branch of each tagged colony. deep-sea organisms. Samples were isolated in individual jars in situ and returned to the A recently discovered shallow-water population of red tree surface where they were kept in containers of cold seawater until corals in Tracy Arm, Holkham Bay, Southeast Alaska provided an onboard processing. The number of polyps per 1 cm was counted unprecedented opportunity to collect reproductive tissue samples for each sample collected, averaging over a 5 cm span of skeleton. at regular intervals year-round from the same colonies. Red tree Ten of the 38 colonies were randomly selected following the corals exhibit keystone species characteristics in Gulf of Alaska initial sampling (September 2010) for the simulated physical habitats where it provides refuge habitat for at least 12 species of damage treatment group. Physical damage consisted of tearing a rockfish ( sp.) including gravid females and juveniles (Stone large branch(s) from each colony that comprised 21–40% of the pers obs), making it an ideal candidate for reproductive study. In total colony volume. This degree of damage is consistent with that September 2010, we began a 16-month study of the sexuality, observed in situ in the Gulf of Alaska (Stone et al., in preparation). reproductive mode and periodicity of P. pacifica from a single Removed tissue was retained, photographed on a scale board on population within Tracy Arm Fjord. We also examined how deck, and the dry weight was obtained in the laboratory. Image J physical damage (simulated fishing damage) might affect repro- (NIH) was used to calculate the flat-plane area of the removed duction in this species. To our knowledge there is no other branches. published study on any cold-water corals species that utilizes seasonal samples collected from the same individuals on a tri- Environmental Monitoring monthly basis, therefore the resolution of this study is significantly A Star-Oddi DST-CTD (mention of trade names or commer- higher than any reproductive information gained to date. cial companies is for identification purposes only and does not imply endorsement by the National Marine Fisheries Service, Materials and Methods NOAA) compact microprocessor-controlled temperature, depth and salinity recorder was attached to one of the tagged colonies Study Site during the first field season (Sept. 2010) and was retrieved during The study site is located in Tracy Arm, Holkham Bay, the final field season (Jan. 2012). The recorder was programmed to Southeast Alaska (Fig. 1). Tracy Arm is a 49-km long, deep (up record measurements every 6 hours and the depth of deployment to 378 m) glacial fjord that terminates in two tide-water glaciers. was 14.9 m. No permissions were required to work at this site. Tides in this During each sampling period a Seabird Electronics Seacat region are semi-diurnal with a tidal range of more than 7 m. The Profiler was used to collect depth, temperature, salinity, and study site consists of graywacke and granodiorite vertical walls density profiles of the water column to a depth of 50–120 m. ranging from high above sea level (the cliff directly above the site is Profiles were collected directly over the primary study site and one ,450 m and the full mountain peak ,750 m) t o the fjord floor at in the center of the fjord. a depth of almost 300 m. Red tree corals (a non-CITES species) A Sontek Argonaut-MD Doppler current meter equipped with were discovered thriving in several shallow water areas of the fjord an internal compass was moored on an untagged colony in March 2006. The study site contains a relatively large thicket (depth = 11 m) near the in-fjord end of the study site. The meter covering a horizontal distance of about 85 m and is located on the was programmed to record average current velocity and direction north side of the fjord approximately 36 km from the terminal every 10 minutes. The instrument was deployed between 10 moraine that forms a sill guarding the fjord entrance to Holkham March 2011 and 3 January 2012. Bay. The study site is approximately 13.4 and 15.4 km from the Sawyer and South Sawyer Glaciers, respectively. Previous Live Observations observations from scuba and remotely operated vehicle operations Two to 5 cm pieces of each tagged coral colony were dissected indicate that the fjord contains several thickets, small patches and live aboard the research vessel during the March 2011 and isolated red tree corals from depths of less than 10 m to at least September 2011 cruises. Oocytes and spermatocysts were 100 m. extracted from polyps using a Leica S8APO stereo microscope,

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Figure 1. Study site in Southeastern Alaska. The study site in Tracy Arm, Holkham Bay, Southeast Alaska (indicated by the solid triangle symbol). The reproductive ecology of 38 Primnoa pacifica colonies were studied at the site between September 2010 and January 2012. doi:10.1371/journal.pone.0090893.g001 observed and photographed (Motic 10 camera attachment, Motic wax for 24 hrs and embedded using standard histological molds. Optical Inc.) for 24 to 48 hrs. Five micron sections were cut for both fecundity (serially every 90 microns) and for oocyte size analysis (five centrally located Histological Processing slides) on a Leica RM2235 rotary microtome. Sections were Eight centimeter pieces of all 38 tagged corals were preserved in stained with Masson’s Trichrome or Haemotoxylin - Eosin (H&E) 10% neutral-buffered formalin for 24–48 hrs and then washed using standard procedures, and permanently coverslipped. Each and transferred to 70% ethanol for long-term storage. From these slide was examined under an Olympus CX21 microscope, with pieces, two sets of three to five polyps were dissected from each Moticam 5 camera (Motic Optical) attachment used to grab specimen for histological analysis. images. Images were analyzed using Image J (NIH). Polyps were decalcified using RDO Rapid Decalcifier (Apex Engineering Products Corporation) until all spicules were Analysis dissolved. These samples were then dehydrated to 100% ethanol, Male to female ratio of the study population was calculated using a graded ethanol series (10–20% increments), and cleared based on the 38 tagged colonies. For each female colony, one using toluene. Samples were then infiltrated in molten paraffin hundred random oocytes were measured per collection (measuring

PLOS ONE | www.plosone.org 3 April 2014 | Volume 9 | Issue 4 | e90893 Red Tree Coral Reproduction only oocytes with nucleus present to avoid measuring the same structure is unknown but it was formed through a thick layer of oocyte twice), and seasonal oocyte-size frequency graphs were epidermal cells and appeared to be protected by a large outer constructed using feret diameters (the calculated diameter as if the sclerite (Fig. 3b). The internal layers of this structure were oocytes were a perfect circle). Mean oocyte-size for each colony thickened, with one side containing a brush border, and the other was also recorded, and three polyps per female had all producing mucus. previtellogenic and vitellogenic oocytes counted for fecundity The study confirmed that the internal anatomy of P. pacifica was estimations. For male colonies one hundred random spermatocysts common to that seen in other gorgonians (Fig. 3c–f). Polyps had a were staged, and seasonal bar charts constructed with percentage short pharynx and short thick tentacles, with a large retractor of each stage per colony. septal muscle, leading to the gastrovascular cavity. Lining the To examine for differences between simulated damage colonies gastrovascular cavity were eight mesenteries, where the gametes and non-damaged colonies, Mann-Whitney U tests were conduct- form, and a prominent siphonoglyph, with long cilla (Fig. 3f). The ed to assess differences in average oocyte size; average fecundity coenenchyme that connects individual polyps together (and to the and average spermatocyst stages. For comparisons, non-damaged axis) was thick, and filled with solenia tubes (Fig. 3e). colonies of similar size to damaged colonies were used. Non- Unidentified crustacean parasites were occasionally observed at parametric Friedman tests (SPSS 2012) were conducted on mean the base of polyps (Fig. 3g). Polyps that contained parasites did not oocyte sizes and fecundity in females and between average contain gametes (or contained few gametes) and so were not spermatocyst stages in males, to test for synchronicity between utilized for reproductive morphology, periodicity and fecundity individuals within and between months. Total monthly mean estimates. These polyps were typically situated next to reproduc- oocyte size was then calculated, and a Pearson product moment tively active polyps without parasites. correlation (SPSS 2012) was used to examine oocyte size correlations and percentage of late-stage spermatocysts with mean Reproductive Morphology monthly temperature from the recorder. Primnoa pacifica is a gonochoric coral, with colonies being either wholly male or female. , either simultaneous or Results sequential, were not found during this study. Gametes were located at the base of polyps in bundles forming off the eight The Fjord Environment mesenteries (Fig. 4a,c), with each gamete being surrounded by The salinity cell on the Star-Oddi recorder failed, possibly due follicle cells that thickened as the gametes developed (Fig. 4d,f). to fouling, during the first month of deployment, therefore only Once fully mature, gametes were found detached from mesenter- temperature data were available for the full length of the study ies, high in the gastrodermal cavity, presumably for release. (Fig. 2.). Average daily temperatures at 15 m depth ranged from Multiple stages of both oocytes and spermatocysts were often 3.14 to 6.33uC over the course of this study. The coldest average present within single polyps. daily temperature was recorded in February 2011 and the warmest Through live dissection, both ripe oocytes and spermatocysts in late November 2011. The general trend was 1) cooling in early (spermaries) were extracted and observed through the stereomi- December and 2) warming beginning in early May. CTD casts croscope (Fig. 4b). Vitellogenic oocytes were light peach in color taken during research cruises confirmed these temperatures at and an indentation of the germinal vesicle was observed on the 15 m depth. Salinity from CTD casts showed a high of 31.41 in periphery of each oocyte (Fig. 4b). Spermatocysts were an opaque March 2011 and a low of 26.86 in September 2011. Dissolved white color and tended to be slightly larger than ripe oocytes oxygen readings were highest in the cooler months (7.66 mg/l in (Fig. 4b). These cysts remained intact for approximately 24 hrs March 2011) and lowest in the warmer months (4.07 mg/l in when incubated in seawater collected at depth and held at ambient December 2010). Water currents were generally bi-directional 21 water temperatures, then began to disintegrate, releasing sperm with the strongest currents (up to 12 cm sec ) out-fjord with a into the water. Fertilization of oocytes was not observed during weaker reciprocal current in-fjord. study. Oocyte sizes were measured for histological analysis. Previtello- Tagged Colonies genic oocytes measured ranged from 20–220 mm feret diameter Of the 38 colonies collected, 20 were females (45–162 cm axial before undergoing vitellogenesis. Vitellogenic oocytes ranged from length), 17 were males (42–151 cm axial length) and one was non- 220 mm to 802 mm. The nucleus started centrally in the reproductive (42 cm axial length). Due to the absence of true previtellogenic oocytes, and moved peripherally as the oocytes gonads in cnidarians, it is impossible to classify the non- ripen, with trophonema becoming apparent in larger oocytes. All reproductive colony as male or female. The sex ratio of this oocytes were surrounded in follicle cells, with vitellogenic oocytes population was therefore 1.17:1 females:males. The average having the thickest surrounding layer. No larvae were identified in number of polyps per centimeter (axial length) was calculated as the histological sections and none were observed anytime during 7.96 (SE 61.22). The largest colony in our study had a this study, though in several dissections unfertilized oocytes were 2 photographed surface area of around 3500 cm , and the smallest observed adhered to the outside of female polyps in the medial 2 colony around 800 cm . area facing the axis. Spermatocysts measured through histology ranged from 500– Internal Morphology 1000 mm in feret diameter. Spermatocysts were surrounded by a Results from this study confirmed that Primnoa pacifica is membrane, creating a sac off the mesentery where sperm form monomorphic, with asexual reproduction occurring by extra- (Fig. 4e). This sac had a thin layer of follicle cells surrounding them tentacular budding along the central gorgonin axis, and sexual throughout their life cycle. Four stages of spermatocysts were reproduction occurring within the autozoids. This morphology is identified – Stage I showed a loose aggregation of tailless similar to that found in most other gorgonians [25]. Polyps were spermatocytes (and presumably spermatogonium); Stage II non-retractile and were heavily armoured in sclerites (Fig. 3a,b). showed more organization of spermatocytes, with a loose lumen Externally located (Fig. 3b,d), a horny collar structure was found being formed; Stage III consisted of organized spermatocytes and on either side of the polyp (2 per polyp). The function of this spermatids around a distinct, but empty lumen; and Stage IV

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Figure 2. Water temperature records from study site. Average daily water temperature measured at a depth of 15 m at the study site in Tracy Arm, Holkham Bay, Southeast Alaska for the period September 2010 to January 2012. Temperatures were recorded every six hours and four measurements were averaged each day. doi:10.1371/journal.pone.0090893.g002 spermatocysts had lumen packed with sperm tails and were often photographed area of each coral colony was calculated using found detached from the mesentery tissue within the gastrovas- ImageJ (as above), thus an approximate number of polyps was cular cavity. calculated per colony. The largest colony in our study had an approximate potential (i.e. ripe, vitellogenic oocytes) fecundity of Simulated Damage 475,000 oocytes, and the smallest female mature colony a Histological analysis showed that 6 of 10 colonies with simulated potential fecundity of 110,000 oocytes. damage were male and 4 were female (with one female and one male being juvenile, with just a few early stage gametes present). Periodicity Image analysis (Image J, NIH) showed that between 21–40% of Oocyte size frequency patterns (Fig. 6) were highly variable the tissue mass was removed from each of these colonies. None of across the population. Size frequency plots suggest that spawning the damaged colonies showed any indications that reproduction is largely asynchronous between individuals in this population. A was affected. In comparison to the undamaged colonies, neither Friedman test was performed between colonies to test for fecundity (U = 6, p#0.05) or oocyte size (U = 2, p#0.05) was asyncronicity, with a Bonferroni correction for multiple compar- affected in females; nor was spermatocyst development in males isons. Mean oocyte sizes were significantly different over the (U = 3, p#0.05). As there was no significant difference between months sampled (X2 = 12.929, p = 0.044), although no pairwise simulated damage colonies and non-damaged colonies, the results comparison was significant, potentially due to the low sample size. from these colonies were pooled with the non-damaged for the Average and maximum oocyte sizes were plotted against total remaining analysis. colony length (Fig. 7a), and slight correlations were seen in both categories (R2 = 0.51 for maximum; R2 = 0.43 for average). Fecundity Fecundity of vitellogenic oocytes appeared to be on more than a Average polyp fecundity for undamaged colonies was 86 yearly cycle (Fig. 5b), though as with oocyte sizes, there was great (SE623) total oocytes, with 69 (SE614) previtellogenic and 17 variation between individuals. Plotting the minimum and maxi- (SE612) vitellogenic. There was a slight trend of increased mum oocyte sizes versus colony length (Fig. 7b) showed a clear fecundity with colony size but the relationship was not significant trend for smaller colonies to produce smaller oocytes up until a (Fig. 5a, R = 0.24377 & R = 0.025923). The smallest reproduc- length of around 80 cm was achieved. tively mature colony was a male at 42 cm length. The

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Figure 4. Reproductive anatomy of red tree coral. Primnoa Figure 3. External and internal anatomy of red tree coral. pacifica reproductive anatomy. a) In situ photograph of open polyps Primnoa pacifica morphology. a) SEM image of three axial polyps; b) showing gametes at base (g), scale ,3 cm; b) Extracted oocytes (vo) SEM image of single axial polyp with horny collar (c) shown; c) and spermatocysts (sp), scale 2 cm; c) Histological section showing Histological image of whole polyp, scale 1000 microns; d) Histological female polyp, scale 1000 microns; d) Histological section of female section showing muscular attachment for horny collar, scale 500 mi- polyp, scale 500 microns; e) Histological section of male polyp, scale crons; e) Histological section showing axis of the colony, scale 200 microns; f) Histological section of spermatocysts, scale 100 microns. 500 microns; f) Histological section showing siphonoglyph, scale (g, gametes; p, polyp; vo, vitellogenic oocytes; sp, spermatocysts; cs, 200 microns; g) Histological section showing parasite at base of polyp, coenchyme; pv, previtellogenic oocytes; f, follicle cells; sp, spermato- scale 1000 microns. (c, horny collar; t, tentacles; g, gastrovascular cavity; cysts; sc, spermatocytes). sm, septal muscle; p, polyp; m, muscle; ax, axis; co, coenenchyme; s, doi:10.1371/journal.pone.0090893.g004 solenia; par, parasite). doi:10.1371/journal.pone.0090893.g003 percentage of late-stage spermatocysts (r = 20.048, P = 0.05) with water temperature. All individual oocyte size frequencies were assessed visually and, after creating combined monthly graphs, were split into individ- Discussion uals of similar patterns (Fig. 8). In each month examined there was a percentage of mature females with vitellogenic oocytes present The goals of this study were to describe the mode and (‘double cohorts’) and a percentage with only previtellogenic periodicity of reproduction for one of the most abundant and oocytes present (‘single cohorts’). The percentage of individuals widespread deep-sea corals in the North Pacific Ocean. We showing single and double cohort traits differed in each month, studied a population of deepwater emerged Primnoa pacifica and in with a higher percentage of mature females in January 2012, and addition assessed whether reproduction in this species is affected the lowest percentage of mature females in September 2011 and by sub-lethal trauma so as to gauge its ability to sustain 2010. This suggests a larger spawning event early in the year than populations threatened by disturbance from fishing activities. We in the fall months. collected two hundred and 28 samples from 38 tagged colonies One hundred random spermatocysts per colony, per sampling over six sequential seasons and examined more than 10,000 period were staged using the same scale as above (Fig. 9a–c). histological sections. This dataset represents one of the more Figure 9 shows the percentage of spermatocysts at each stage in extensive reproductive studies for any cold-water coral species in undamaged colonies (a); simulated damage colonies (b); and all the world, and provides higher resolution (i.e. same colonies male colonies (c). In both years, September appeared to have the resampled over time) data than previously collected to our highest percentage of late stage spermatocysts, while June and knowledge. September 2011 had the lowest percentages of early-stage Mass spawning events are a common trait amongst broadcast spermatocysts. In all colonies measured, June 2011 had no late- spawning corals, providing for greater genetic diversity, protection stage spermatocysts present in polyps. For males, September 2011 from over-predation and enhancing fertilization success was removed from statistics because no ‘non-damaged’ males were [26,27,28]. In zooxanthellate and azooxanthellate octocorals mass collected that month. spawning events have been tied to temperature, lunar cycles and There was also no correlation between mean oocyte sizes food availability [29,8,30,11,31]. This population of P. pacifica does (r = 0.205, P = 0.05), mean fecundity (r = 0.077, P = 0.05), or the not appear to fit with either a mass spawning event, or quasi- continuous spawning events documented in other octocorals

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Figure 5. Fecundity measures for red tree coral. A) Size specific fecundity for Primnoa pacifica colonies. Mean total fecundity (dark) averaged over the six time points, and June mean fecundity (open) used. Neither measurements show a positive correlation with axial length; B) Mean total fecundity over months analyzed for seven individuals showing varied patterns per colony. doi:10.1371/journal.pone.0090893.g005

Figure 6. Seasonal oocyte size frequency graphs. Examples of seasonal oocyte size frequency graphs for four individual colonies collected in this study. doi:10.1371/journal.pone.0090893.g006

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Figure 7. Oocyte size analysis. A) Range of oocyte sizes plotted against length of colony. Logarithmic trendlines are shown; B) Maximum and minimum oocyte sizes plotted against colony length. doi:10.1371/journal.pone.0090893.g007

[32,33,11,34] or scleractinians (see review in [28]). Intra-colony strategies for energy storage [40], we can hypothesize that this variation was not observed in this study (i.e. all polyps appeared to population of P. pacifica, with its ability to reproduce with marked have similar gamete stages and fecundity); however, there was inter-colony asynchronicity, permanent availability of previtello- great asynchronicity of inter-colony gametogenesis and apparent genic oocytes for maturation and highly seasonal food availability, spawning events. Some females appeared to mature oocytes twice would be efficient at both storing and potentially quickly utilizing a year, whereas others only matured oocytes once over the 16 food resources for reproductive purposes. Studies into energy months sampled or appeared to have a continuous supply of investment and storage in this species would be highly valuable to mature oocytes (Fig. 6 and Fig. S2). Duration of gametogenesis understanding the wider implications of their high reproductive tends to be ‘long’ in many octocoral species, primarily because outputs, and their ability to sustain populations in both deep and larger, energetically expensive, oocytes tend to be the norm [35– shallow ecosystems. 37]. Primnoa resedaeformis from the North Atlantic appear to have all These data provide important and timely insights regarding the stages of oogenesis in each polyp, suggesting overlapping cohorts capability of some of the region’s dominant benthic epifauna to or quasi-continuous oogenesis in this closely related species [38]. recover from disturbance and to recolonize areas previously No males were observed in that study but Mercier et al. [38] also disturbed by human activities. The recovery rate of disturbed found that oocyte sizes were similar (though larger, at 1000 mi- benthic ecosystems in the Gulf of Alaska is of keen interest to crons) as was fecundity and reproductive mode (i.e. no larvae were fisheries managers given recent actions in the region to preserve observed), showing potential conservation of traits amongst the areas of known coral abundance [44], including P. pacifica thickets genus [34]. [4]. Large Primnoa colonies (.2 m height) are rare in the Gulf of Male colonies did appear to show some periodicity to sperm Alaska and are much more susceptible to damage from fishing maturation (Fig. 9), with potentially three spawning periods gear. In contrast to what has been shown in several other studies observed during this study – September 2010 through December (e.g. [45,46,47,12,13]), our comparison of undamaged and 2010, March 2011 through June 2011, and again between damaged colonies did not indicate any adverse effects to September 2011 and January 2012. At the beginning of the reproduction from sub-lethal physical trauma. However, sub- study, in September 2010, male colonies had all four stages of lethal trauma, like that simulated in this experiment, reduces the spermatocysts present in nearly equal percentages. By December overall fecundity of colonies in direct relation to the amount of the 2010 many of the late-stage spermatocysts had disappeared, colony that is removed. presumably through spawning (though this was not observed), and Our results also indicate that colonies smaller than 42 cm in more early-stage spermatocysts were being developed. In March axial length are non-reproductive and that female colonies smaller 2011 many of these early stages had developed into Stage IV than ,80 cm produce smaller oocytes than larger colonies. spermatocysts, which were then spawned off by the June 2011 Oocytes are energetically expensive to produce, particularly the collection. Then in September 2011 (with the caveat this was a low larger (,300–1000 mm) yolky eggs that appear to be characteristic collection season) earlier stage spermatocysts appear to quickly of octocorals [36]. Oocyte size can influence fertilization success in ripen for spawning through January 2012. Although there are marine invertebrates, with sperm availability influencing optimal three spawning cycles observed in this study (two within a single oocyte sizes for individual species [48]. In this population, smaller year), the percentages of early-stage spermatocysts available for colonies produced smaller oocytes than larger colonies, creating a development appeared to have their own pattern. This cycle dichotomy of oocytes sizes within a single population. Though appeared to take just over a year to complete – early development there have been some reports of different populations having occurred through September 2010, and large numbers were not different oocyte sizes (creating genetic subdivision, [49]); these regenerated again until somewhere between September 2011 and differences appear rare [48] and we could find no reports of January 2012. different oocyte sizes within a single population. In urchins, some Although spawning periods for males and females might occur species of Strongylocentrotus with smaller oocytes require a higher throughout the year, corals may only be able to produce germ cells concentration of sperm to fertilize at equal rates to species with for spermatogenesis on a yearly cycle, and may only be able to larger oocyte sizes, though species from a different genus show no mature oocytes through vitellogenesis sporadically, maybe when differences (reviewed in [48]). For this population from Tracy Arm conditions are suitable for spawning. The trophic ecology of this fjord, if larger colonies produce the ‘optimal’ size of oocyte for the species is unknown, but there are ties between food availability, relative sperm availability and adaptation, then oocytes produced lipid synthesis and reproduction in cold-water octocoral species by smaller colonies could have less fertilization success than those [39,40]. This temperate ecosystem is strongly influenced by of larger size. This could have an overall effect on total population seasonal pulses of plankton but given the steep fjord wall fecundity should colonies be broken to smaller size classes through environment the corals inhabit, they are not able to rely on the fisheries damage. predictable ‘food bank’ afforded to other temperate and polar Given the long duration of gamete development and seemingly communities inhabiting horizontal seafloor to continue reproduc- long recruitment times (i.e. no new recruits were observed in this tion year-round [41,42]. The study area is subjected by a strong study area, pers. obs), removal of polyps able to produce germ cells predictable spring phytoplankton bloom each year, with minor and mature gametes would be directly detrimental to population subsequent blooms during the summer months [43]. We observed sustainability. Removal of entire colonies is lethal and effectively no obvious coupling of reproductive events for corals in this study removes the entire reproductive output for those colonies and and the timing (April) of the spring phytoplankton bloom and results from this study indicate that recruitment events are likely to therefore conclude that the timing of reproduction in this species is be highly sporadic in this species. In addition polyp level fecundity controlled by other factors, possibly a complex suite of environ- has been found to be related to overall population density in other mental factors. Though different octocoral species exhibit different cnidarians including gorgonians and pennatulids [36]. Discrete

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Figure 8. Seasonal oocyte size frequency graphs. Oocyte size frequency data – Combined - all females with oocytes present averaged; Single – all females with only previtellogenic oocytes; Double – all females with both previtellogenic and vitellogenic oocytes. N = number of colonies combined. doi:10.1371/journal.pone.0090893.g008

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Figure 9. Seasonal male analysis. Stacked bar graph showing stages of spermatogenesis over sampling periods analyzed. a) Non damaged colonies; b) simulated damage colonies; c) combined means. doi:10.1371/journal.pone.0090893.g009 areas of with abundant, large P. pacifica colonies would be good it has been found in other species that though sexuality and candidates for reproductive refugia such as the Habitat Areas of reproductive mode is constrained, fecundity can be reduced Particular Concern (HAPCs) that were designated by the North [52,38,53]. The results from this study will provide for a Pacific Fishery Management Council in 2007 [3,4]. comparison with samples presently being collected from deep- Primnoidae are the most abundant corals in Alaskan waters water so that we can address questions regarding whether depth- [50]. These findings might be applicable to the other 18 species in related ecological effects occur in this species, and aid in forming this family found in Alaskan waters. Kahng et. al. [34] reported management plans for those specific populations. that sexuality and reproductive mode tend to be conserved in families amongst octocorals, with gonochorism and internal Supporting Information brooding being dominant traits in the Primnoidae. The results of this study confirmed that P. pacifica is indeed a gonochoric Figure S1 Graph showing colony lengths against depth species but it not an internal brooder. We found on several females of colony. Graph shows colonies selected covered a wide variety polyps where unfertilized oocytes were ‘‘stuck’’ to the epidermis of of sizes between our defined depth range; male and female the polyps in a central area. We also question the purpose of the colonies were spread out with respect to depth; and that colonies ‘‘collar’’ structure. This thickened epiderm is potentially lined with selected for simulated damage were selected haphazardly around a brush border, filled with mucus and protected by sclerites, the sample site. There is a non-significant trend towards larger making it an ideal protected environment for external brooding. colonies being deeper at this site (R2 = 0.434). Decalcification for histology would loosen this areas hold on (TIF) gametes or larvae, potentially losing firm identification of this Figure S2 Graphs of individual oocyte size frequency structures function. Only further studies through electron micros- data. Each graph is for a single individual with each month of copy might elucidate the function of this structure. data plotted. (TIF) Conclusions This study focused on an accessible shallow-water population of Acknowledgments P. pacifica, yet this species is more commonly found in deep-water (125 to 800 m) in the Gulf of Alaska. The purpose of this study was We’d like to acknowledge the help and support of Captain Dennis Rogers and the crew of the MV Alaskan Legend and MV Northern Song and Captain to provide baseline data from which to compare deep-sea samples Dan Foley and crew of the MV Steller. We thank Brad Weinlaeder and in the future, as well as provide information to evaluate the Christian Clark for helping us in the field. Kevin Eckelbarger provided potential effectiveness of management measures for this species valuable assistance in histology interpretation of new structures, and such as designation of Habitat Areas of Particular Concern [3,4]. Michele Masuda helped with statistical analyses. Keri Feehan and Maggie In this shallow water situation, P. pacifica shows a spawning Halfman were essential in preparing and analyzing histological samples reproductive mode, with oocyte and spermatocyst development and we thank Laura Grange for providing helpful comments on this generally taking over a year for a full cycle. Though spermatocyst manuscript. The findings and conclusions in the paper are those of the development appears synchronous, oocyte development is highly author(s) and do not necessarily represent the views of the National Marine asynchronous with multiple spawning events occurring during a Fisheries Service. single year (2–3 events observed during this study). This strategy would allow for enhanced genetic diversity, and, for a population Author Contributions living at the extreme edge of the species bathymetric range, within Conceived and designed the experiments: RGW RPS JM. Performed the generation bet-hedging on when in the year optimum larval experiments: RGW RPS JM JJ. Analyzed the data: RGW JJ RPS. development, survival and recruitment would occur [51]. In the Contributed reagents/materials/analysis tools: RGW. Wrote the paper: deep ocean, where seasonal cues can be more discrete and limited, RGW RPS JM JJ.

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